Fig. 7. Transient transfection studies with exogenous Src corroborate its role in SHIP2 phosphorylation. (A) IPs (anti-SHIP2 and pre-immune) from HeLa cells transiently transfected with expression constructs encoding constitutively active Src (CA-Src) or dominant-negative Src (DN-Src) were blotted with anti-phosphotyroisne (-PY). Mock-transfected cells were included as a control. Cells were either adherent on plastic (Ad), detached (D) or freshly re-plated on collagen-I-coated surface (6 µg/cm²) for 60 minutes. The arrow points to tyrosine-phosphorylated SHIP2. The bottom panel shows an anti-SHIP2 blot of the above immunoprecipitate samples. (B) Whole cell lysates prepared from the cells transfected as described above were blotted with anti-Src antibody. (C) Densitometric analyses of SHIP2 tyrosine phosphorylation levels observed in mock-transfected or DN-Src-transfected HeLa cells that were re-plated on a collagen-I-coated surface (6 µg/cm²) for 60 minutes.